Difference between revisions of "Ovoperoxidase from Sea Urchins"
| (28 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
==Project Overview== | ==Project Overview== | ||
| − | The main goal of this project is to isolate the sea urchin ovoperoxidase enzyme and collect data about it's activity and reactions with hydrogen peroxide. Former student Laura Sanchez was isolating the protein, but upon graduation her work was passed down to Emily Rein who will continue to work on isolating the enzyme for our use. | + | The main goal of this project is to isolate the sea urchin ovoperoxidase enzyme and collect data about it's activity and reactions with hydrogen peroxide. Former student Laura Sanchez was isolating the protein, but upon graduation her work was passed down to Emily Rein who will continue to work on isolating the enzyme for our use. Another student, [[William_B_Fox|Will Fox,]] will be assisting with the synthesis and analysis of the sperm peroxidase. |
The following video portrays the actual fertilization process for sea urchins: | The following video portrays the actual fertilization process for sea urchins: | ||
| Line 23: | Line 23: | ||
File:SU_egg_01.jpg|Sea Urchin Egg (02/27/2019) | File:SU_egg_01.jpg|Sea Urchin Egg (02/27/2019) | ||
File:SU_egg_02.jpg|Enhanced Image of Sea Urchin Egg (02/27/2019) | File:SU_egg_02.jpg|Enhanced Image of Sea Urchin Egg (02/27/2019) | ||
| + | File:single_su_egg_070919.png|Enhanced Image of a Single Sea Urchin Egg (short spined) (07/09/2019) | ||
| + | File:su_eggs_070919.png|Enhanced Image of Sea Urchin Eggs (short spined) (07/09/2019) | ||
| + | File:su_eggs_2_070919.png|Enhanced Image of Sea Urchin Eggs (short spined) (07/09/2019) | ||
| + | File:su_eggs_3_070919.png|Enhanced Image of Sea Urchin Eggs (short spined) (07/09/2019) | ||
</gallery> | </gallery> | ||
| + | |||
| + | ==Ovoperoxidase== | ||
| + | |||
| + | Ovoperoxidase is an enzyme, released from eggs at fertilization, that creates a fertilization envelope around the sea urchin egg by to prevent multiple sperm from fertilizing the egg. If multiple sperm reach the egg, the egg will not become fertilized. This hardening process is due to the oxidation of protein-bound tyrosyl residues, which happens in the presence of a peroxidase. | ||
| + | |||
| + | ==Sea Urchin Sperm Peroxidase== | ||
| + | |||
| + | When a Sea Urchin egg is fertilized, it releases hydrogen peroxide into the surrounding space. This peroxide interacts with a peroxidase endogenous to the sperm to render the sperm infertile, and in doing so helps prevent polyspermy. | ||
| + | |||
| + | ==Preparation== | ||
| + | In preparation for the egg extraction, sea water was made with a specific gravity of 1.015, which was measured with a refractometer. Some of this sea water was combined with the water the sea urchins came in and poured in an aquarium with a 50/50 mixture. This aquarium is where the urchins were placed after they were done releasing their gametes. The rest of the sea water was saved for use in the cups the urchins were placed in to release gametes. | ||
==Egg Extraction== | ==Egg Extraction== | ||
| Line 34: | Line 49: | ||
[https://www.youtube.com/watch?v=VZTqVPe8alM In Lab Video] | [https://www.youtube.com/watch?v=VZTqVPe8alM In Lab Video] | ||
| + | |||
| + | '''July 9, 2019''' | ||
| + | |||
| + | For the second round of sea urchins, 1 mL of 0.55 M KCl was injected into the sea urchins. This round had more females, but most of the sea urchins arrived dead or ill. We had 5 females produce eggs and 4 males produce sperm. | ||
| + | |||
| + | To prevent cross-contamination of eggs and sperm, each urchin was taken out of the bag, placed mouth-side up on a new sheet of aluminum foil, injected, and then placed mouth-side up into its own plastic cup filled with sea water. | ||
===Our Lab Experience=== | ===Our Lab Experience=== | ||
| Line 44: | Line 65: | ||
File:IMG_1112.JPG|Sea urchin eggs | File:IMG_1112.JPG|Sea urchin eggs | ||
File:IMG_1114.JPG|Sea urchin spine | File:IMG_1114.JPG|Sea urchin spine | ||
| + | File:seaurchinscups.jpg|Short spined sea urchins | ||
File:RenderedImage (1).JPG|Sea urchin egg | File:RenderedImage (1).JPG|Sea urchin egg | ||
File:RenderedImage.JPG|Magnified sea urchin egg | File:RenderedImage.JPG|Magnified sea urchin egg | ||
| Line 49: | Line 71: | ||
==Instrumentation== | ==Instrumentation== | ||
| − | The UV-Vis is the main instrument we use to analyze the peroxidase. | + | The UV-Vis is the main instrument we use to analyze the activity peroxidase. |
The following link explains the procedure used with the UV-Vis. | The following link explains the procedure used with the UV-Vis. | ||
[[UV_Vis|UV-Vis]] | [[UV_Vis|UV-Vis]] | ||
| + | |||
| + | ==Data Analysis== | ||
| + | |||
| + | The data we collected from the UV-Vis is analyzed in IGOR. | ||
| + | |||
| + | The following link explains how to use IGOR to analyze this data. | ||
| + | |||
| + | [[IGOR|IGOR]] | ||
==References== | ==References== | ||
| Line 61: | Line 91: | ||
[[Sea_Urchin_Fertilization_Lab|Sea Urchin Fertilization Lab - Brad's Page]] | [[Sea_Urchin_Fertilization_Lab|Sea Urchin Fertilization Lab - Brad's Page]] | ||
| + | |||
| + | [https://www.pnas.org/content/pnas/74/10/4214.full.pdf Tyrosine Crosslinks] | ||
| + | |||
| + | [[:File:Sea_urchin_sperm_peroxidase_is_competitively_inhib.pdf| Sea Urchin Sperm Peroxidase]] | ||
==Scots J-Term Andros Island== | ==Scots J-Term Andros Island== | ||
| − | January 4th through January 12th of 2019 my research advisor, Brad Sturgeon, along with | + | January 4th through January 12th of 2019 my research advisor, Brad Sturgeon, along with seven other students and myself, visited Forfar Field Station on Andros Island in the Bahamas. There I was able to learn about the natural environment of sea urchins, as well as other relevant science such as geology. We also gained field experience by studying them through snorkeling. The interns at Forfar were very knowledgeable about the area and the creatures inhabiting the ocean around the island. Emily Rein and Will Fox, who are involved in different aspects of the sea urchin research, were among the seven students on the trip. |
Below is a link to an article written about the trip as a whole: | Below is a link to an article written about the trip as a whole: | ||
[http://monmouthcollege.meritpages.com/news/monmouth-college-students--explore-new-worlds--on-one-week-academic-trip-to-the-bahamas/7194?fbclid=IwAR3JOLqcFYNKls6f7rsYAbn1_zJ195XaUMS-ev_IjFWNxHeGl177VBKaLY8| Bahamas Trip] | [http://monmouthcollege.meritpages.com/news/monmouth-college-students--explore-new-worlds--on-one-week-academic-trip-to-the-bahamas/7194?fbclid=IwAR3JOLqcFYNKls6f7rsYAbn1_zJ195XaUMS-ev_IjFWNxHeGl177VBKaLY8| Bahamas Trip] | ||
Latest revision as of 20:03, 30 June 2020
Project Overview
The main goal of this project is to isolate the sea urchin ovoperoxidase enzyme and collect data about it's activity and reactions with hydrogen peroxide. Former student Laura Sanchez was isolating the protein, but upon graduation her work was passed down to Emily Rein who will continue to work on isolating the enzyme for our use. Another student, Will Fox, will be assisting with the synthesis and analysis of the sperm peroxidase.
The following video portrays the actual fertilization process for sea urchins:
The following link shows the anatomy of a sea urchin:
The Sea Urchins
Gulf Specimen Marine Laboratories is the vendor through which we purchased the sea urchins for research. We purchased around 12 fertilized sea urchins, and were able to collect eggs from one sea urchin out of the first batch.
Below is a link to the website from which we ordered the sea urchins:
Gulf Specimen Marine Laboratories
The following images are of the sea urchin eggs we abstracted.
Sea Urchin Egg (02/27/2019)
Enhanced Image of Sea Urchin Egg (02/27/2019)
Enhanced Image of a Single Sea Urchin Egg (short spined) (07/09/2019)
Enhanced Image of Sea Urchin Eggs (short spined) (07/09/2019)
Enhanced Image of Sea Urchin Eggs (short spined) (07/09/2019)
Enhanced Image of Sea Urchin Eggs (short spined) (07/09/2019)
Ovoperoxidase
Ovoperoxidase is an enzyme, released from eggs at fertilization, that creates a fertilization envelope around the sea urchin egg by to prevent multiple sperm from fertilizing the egg. If multiple sperm reach the egg, the egg will not become fertilized. This hardening process is due to the oxidation of protein-bound tyrosyl residues, which happens in the presence of a peroxidase.
Sea Urchin Sperm Peroxidase
When a Sea Urchin egg is fertilized, it releases hydrogen peroxide into the surrounding space. This peroxide interacts with a peroxidase endogenous to the sperm to render the sperm infertile, and in doing so helps prevent polyspermy.
Preparation
In preparation for the egg extraction, sea water was made with a specific gravity of 1.015, which was measured with a refractometer. Some of this sea water was combined with the water the sea urchins came in and poured in an aquarium with a 50/50 mixture. This aquarium is where the urchins were placed after they were done releasing their gametes. The rest of the sea water was saved for use in the cups the urchins were placed in to release gametes.
Egg Extraction
0.5 mL of 0.55 M KCl was injected into the sea urchins to cause them to release their eggs/sperm. 0.25 mL was injected into either side of the mouth. After the KCl was injected, the urchins were overturned on a beaker to release the eggs/sperm.
The first round of sea urchins had only 4 females.
The following video was used as references to guide our lab experience.
July 9, 2019
For the second round of sea urchins, 1 mL of 0.55 M KCl was injected into the sea urchins. This round had more females, but most of the sea urchins arrived dead or ill. We had 5 females produce eggs and 4 males produce sperm.
To prevent cross-contamination of eggs and sperm, each urchin was taken out of the bag, placed mouth-side up on a new sheet of aluminum foil, injected, and then placed mouth-side up into its own plastic cup filled with sea water.
Our Lab Experience
Injecting the urchins with KCl
A sea urchin releasing sperm
Sea urchin sperm release
Sea urchin sperm release
A sea urchin releasing eggs
Sea urchin eggs
Sea urchin spine
Short spined sea urchins
Sea urchin egg
Magnified sea urchin egg
.JPG)
Instrumentation
The UV-Vis is the main instrument we use to analyze the activity peroxidase.
The following link explains the procedure used with the UV-Vis.
Data Analysis
The data we collected from the UV-Vis is analyzed in IGOR.
The following link explains how to use IGOR to analyze this data.
References
Sea Urchin Fertilization Lab - Brad's Page
Scots J-Term Andros Island
January 4th through January 12th of 2019 my research advisor, Brad Sturgeon, along with seven other students and myself, visited Forfar Field Station on Andros Island in the Bahamas. There I was able to learn about the natural environment of sea urchins, as well as other relevant science such as geology. We also gained field experience by studying them through snorkeling. The interns at Forfar were very knowledgeable about the area and the creatures inhabiting the ocean around the island. Emily Rein and Will Fox, who are involved in different aspects of the sea urchin research, were among the seven students on the trip.
Below is a link to an article written about the trip as a whole: